WO2019094265A1

WO2019094265A1 - Pd1 polypeptide binding molecules

Info

Publication number: WO2019094265A1
Application number: PCT/US2018/058824
Authority: WO
Inventors: Scott Mccauley
Original assignee: Armo BioSciences Inc
Current assignee: Armo BioSciences Inc
Priority date: 2017-11-10
Filing date: 2018-11-02
Publication date: 2019-05-16
Anticipated expiration: 2020-05-10

Abstract

The present disclosure provides polypeptide binding molecules that bind to Programmed Cell Death-1 (PD-1) and inhibit PD-1 interaction with both Programmed Cell Death Ligand-1 (PD-L1) and/or Programmed Cell Death Ligand-2 (PD-L2). The present disclosure further provides pharmaceutical formulations comprising the molecules of the present invention. The present disclosure further provides methods of use of such polypeptide binding molecules in the treatment of various disorders and diseases. The present disclosure further provides recombinant nucleic acid sequences encoding such polypeptide binding molecules and cells transformed therewith. The present disclosure also provides methods of use of the molecules of the present invention in the diagnosis, prevention and treatment of various disorders or conditions in subjects including but not limited to the treatment of neoplastic disorders including but not limited to cancer.

Description

Title

PD1 Polypeptide Binding Molecules Introduction

[0001] PD-1 is a 288 amino acid protein expressed in B, T and myeloid derived cells.

The cDNA of PD-1 was isolated in 1992 from a murine T cell hybridoma and a hematopoietic progenitor cell line undergoing apoptosis. It has become clear over the last 24 years that PD-l 's primary function is to attenuate immune responses. For example, PD-l 's role in controlling the immune system was revealed by genetic ablation studies where PD-1 deficiency resulted in multiple autoimmune phenotypes in different murine backgrounds. PD-1 has been shown to negatively regulate responses to antigenic stimulation. This was elucidated using allogeneic T cells expressing transgenic T cell receptors. In the absence of PD-1, these T cells exhibited enhanced responses to alloantigen stimulation.

[0002] PD-1 binds to both PD-L1 (B7-H1) and PD-L2 (B7-DC). The interaction of PD-1 with its ligands mediates its inhibitory effects. PD-L2 expression is limited to professional antigen presenting cells, whereas PD-L1 is expressed over a much broader tissue distribution. Interactions between PD-L1 and PD-L2 are thought to maintain peripheral tolerance.

[0003] Due to the development of auto-immunities in PD-1 deficient mice, inhibition of the PD-1 pathway was not automatically justified for use in cancer immunotherapy. However, while it was discovered that PD-L1 message and protein are expressed by many fresh human tumor samples, PD-L1 protein is normally only expressed in tonsil, in tissue associated macrophages in the lung and liver, and placenta. In particular, PD-L1 is very rarely expressed in human tumor cell lines cultured in vitro. The discrepancy between fresh human tumor expression and in vitro cultured tumor cell line expression of PD-L1 was explained by the findings that ΠΤΝΓγ upregulates the PD-1/PD-L1 pathway. These findings have led to the adaptive resistance hypothesis. This hypothesis posits that as tumor antigen specific CD8+ T cells respond to MHC I presented tumor antigens, the T cells will secrete IFNy at the interface between the T cell and the target cell. ΠΤΝΓγ will then induce the upregulation of both MHC I/antigen, and the PD-1/PD-L1 pathway. Simultaneous expression of MHC I/antigen complexes and the PD-1/PD-L1 pathway functionally paralyzes the responding CD8+ T cells, leading to peripheral tolerance.

[0004] The United States Food and Drug Administration has approved two anti-PD-1 monoclonal antibodies for the treatment of human cancers, OPDIVO® (nivolumab, BristolMyers Squibb, Princeton NJ) and KEYTRUDA® (pembrolizumab, Merck & Co., Kenilworth NJ). OPDIVO® was the first mAb targeting PD-1 to show significant clinical activity in unresectable or metastatic melanomas, non-small-cell lung carcinoma (NSCLC), and metastatic renal cell carcinoma. OPDIVO® has consistently induced objective response rates (ORR) of 30-40% in multiple clinical trials. KEYTRUDA® has exhibited similar efficacy and safety to OPDIVO® in a Phase 1 clinical trial and is now an FDA-approved second-line drug for the treatment of melanoma.

[0005] The present disclosure provides polypeptide binding molecules that bind to

Programmed Cell Death-l 's ("PD-1" or "PD1") and inhibit interaction of PD-1 with both Programmed Cell Death Ligand-1 (PD-L1) and/or Programmed Cell Death Ligand-2 (PD-L2).

[0006] The present disclosure further provides compositions useful in the preparation of such polypeptide binding molecules including nucleic acid sequences encoding said polypeptide binding molecules and recombinant cell lines expressing said polypeptide binding molecules [0007] The present disclosure further provides pharmaceutical formulations comprising the molecules of the present invention.

[0008] The present disclosure further provides methods of use of such polypeptide binding molecules in the treatment of various disorders and diseases. The present disclosure further provides recombinant nucleic acid sequences encoding such polypeptide binding molecules and cells transformed therewith.

[0009] The present disclosure also provides methods of use of the molecules of the present disclosure in the diagnosis, prevention and treatment of various disorders or conditions in subjects including but not limited to the treatment of neoplastic disorders including but not limited to cancer.

Brief Description of the Figures:

[0010] Figure 1 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 2 of the present disclosure demonstrating that AMOOOl 's inhibition of PD-1 :PD-L1 is comparable to that of both

OPDIVO® (nivolumab) and KEYTRUDA® (pembrolizumab)

[0011] Figure 2 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 4 of the present disclosure demonstrating that AMOOOl blocks PDL1 binding

[0012] Figure 3 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 5 of the present disclosure demonstrating AMOOOl binding specificity for human, cynomolgus, and murine PD-1

[0013] Figure 4 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 5 of the present disclosure demonstrating AMOOOl binding specificity for Rat PD-1

[0014] Figure 5 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 5 of the present disclosure demonstrating AMOOOl binding specificity for Canine PD-1.

[0015] Figure 6 of the attached drawings provides an illustration of a gel stained with

Coomassie blue demonstrating effective deglycosylation of the PD-1 substrate under the reaction conditions as provided in Example 6 of the present disclosure.

[0016] Figure 7 of the attached drawings provides comparative binding data of AMOOOl and nivolumab to glycosylated and deglycosylated forms of the human and mouse PD-1 proteins under reaction conditions as provided in Example 7. [0017] Figure 8 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 6 of the present disclosure demonstrating AM0001 blocks PD-1 binding to PD-L1 and PD-L2

[0018] Figure 9 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 7 of the present disclosure demonstrating the ability of AM0001 to inhibit of endogenous PD1 function in vitro.

Detailed Description of the Invention

I. General/Intro:

[0019] Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Where a range of values is provided, it is understood (unless the context clearly dictates otherwise) that each intervening value to the tenth of the unit between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention as if explicitly stated. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Headings are provided only for the convenience of the reader and are not to be understood as exclusive in relation to the subject header information.

II. Definitions

[0020] Unless otherwise indicated, the following terms are intended to have the meaning set forth below. Other terms are defined elsewhere throughout the specification. Unless defined otherwise, technical and scientific terms used herein shall be construed as having the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] Activity: The term "activity," in reference to an PDl-PBM ("PD-1 Polypeptide

Binding Molecule) of the present disclosure, includes, but is not limited to, epitope or antigen binding affinity, ability to neutralize or antagonize a bioactivitv of PD-1 in vivo or in vitro and/or in vitro stability of an immunoglobulin (e.g. antibody) and the immunogenic properties of the immunoglobulin (e.g., antibody), e.g., when administered to a human subject. The

aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, ELISA, competitive ELISA, surface plasm on resonance analysis, in receptor binding and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as the need may be.

[0022] Affinity: The term "affinity" as used herein refers to the degree of specific binding of a PBM (e.g., immunoglobulin) to its target and is measured by the binding kinetics expressed as K_d, a ratio of the dissociation constant between the PDl-PBM and the extracellular domain of PDl (K₀ff) and the association constant between the PBM and the extracellular domain of PDl (Kon). As used herein, the term "high affinity" is used in reference to PDl-PBMs having a Kd<10e-07 , alternatively Kd<10e-08, alternatively Kd<10e-09 as determined by surface plasmon resonance (e.g. Biacore). In one embodiment, PDl-PBM of the present disclosure have a Kd for cynomologous monkey PD-1 of about 100 pM or less at 25^C'C. In further embodiments, PDl-PBMs of the present disclosure have a binding affinity for human and cynomologous PD-1 of about 10 pM or less at 25°C and a binding affinity for cynomologous monkey PD-1 of about 100 pM or less at 25°C,

[0023] Comparable: As used herein, the terms "comparable", "comparable activity",

"activity comparable to", "comparable effect", "effect comparable to", are understood as relative terms that can be viewed quantitatively and/or qualitatively. When comparing one result to another result (e.g., one result to a reference standard^ "comparable" frequently means that one result deviates from a reference standard by less than 35%, by less than 30%, by less than 25%, by less than 20%, by less than 15%, by less than 10%, by less than 7%, by less than 5%, by less than 4%, by less than 3%, by less than 2%, or by less than 1%. In particular embodiments, one result is comparable to a reference standard if it deviates by less than 15%, by less than 10%, or by less than 5% from the reference standard.

[0024] Derived From : The term "derived from" as used herein in the context of a polypeptide sequence or polynucleotide sequence based on a reference polypeptide or nucleic acid without regard to the method of generation of such polypeptide sequence or polynucleotide sequence. Homologs and variants are examples of polypeptide sequences or nucleic acid or nucleic acid sequences that are derived from a reference polypeptide or nucleic acid.

[0025] Enriched: The term "enriched" refers to a sample is non-naturally manipulated so that component (e.g. a polypeptide) is present in: a) a greater concentration (e.g., at least 3 -fold greater, at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or more) than the concentration of the component in the starting sample, or b) a concentration greater than the environment in which the component was made.

[0026] Inhibit/Neutralize: The term "inhibit" or "neutralize" as used herein with respect to a bioactivity of an PD1-PBM of the present disclosure means the ability to substantially antagonize, inhibit prevent, restrain, slow, disrupt, eliminate, stop, reduce or reverse a bioactivity of PD-1 (e.g., as measured in Example 3 herein).

[0027] Isolated: The term "isolated" is used in reference to a polypeptide or nucleic acid of interest that, if naturally occurring, is in an environment different from that in which it can naturally occur. "Isolated" is meant to include polypeptides or nucleic acids that are within samples that are substantially enriched for the polypeptide or nucleic acid of interest and/or in which the polypeptide or nucleic acid of interest is partially or substantially purified. Where the polypeptide or nucleic acid is not naturally occurring, "isolated" indicates that the polypeptide or nucleic acid has been separated from an environment in which it was made by either synthetic or recombinant means.

[0028] CDRs. As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services,

"Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); by Chothia et al., J. Mol. Biol. 196:901 -917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues, which encompass the CDRs, as defined by each of the above cited references are set forth below in Table 1 as a comparison.

Table 1: CDR Definitions

Residue numbering follows the nomenclature of Kabat et al., supra Residue numbering follows the nomenclature of Chothia et al., supra Residue numbering follows the nomenclature of MacCallum et al., supra

[0029] Kabat Numbering The term "Kabat numbering" as used herein is recognized in the art and refers to a system of numbering amino acid residues which are more variable (e.g., hypervariable) than other amino acid residues in the heavy and light chain regions of

immunoglobulins (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 ( 1971); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91 -3242 ( 1991)). For purposes of the present disclosure, the positioning of CDRs in the variable region of an antibody follows Kabat numbering or simply, "Kabat."

[0030] Modulator: The term "modulator" is meant to refer broadly agents that directly or indirectly increase or decrease the function or activity of an PDl-PBM. Examples of modulators include chemical compounds or physical phenomena (e.g., radiation). The terms "modulate", "modulation" refer to the effect of a modulator.

[0031] N-Terminal/C-Terminal: As used herein in the context of the structure of a polypeptide, "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms "N-terminal" and "C-terminal" refer to relative positions in the amino acid sequence of the polypeptide toward the N-terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C-terminus, respectively. "Immediately N-terminal" or

"immediately C-terminal" refers to a position of a first amino acid residue relative to a second amino acid residue where the first and second amino acid residues are covalently bound to provide a contiguous amino acid sequence.

[0032] Nucleic Acid: As used herein the terms "nucleic acid," "nucleic acid", "nucleic acid molecule", "polynucleotide" and the like are used interchangeably herein to refer to a phosphodiester-linked polymeric form of nucleotides of any length, either deoxy-ribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers and the like.

[0033] PD-1 : As used herein, the term "PD-1 "(or "PD1") refers to the 288 amino acid polypeptide having the amino acid sequence:

MQI PQAPWPV VWAVLQLGWR PGWFLDS PDR PWNPPT FS PA LLVVTEGDNA T FTCS FSNT S ES FVLNWYRM S PSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAI SLAP KAQI KE SLRA ELRVTERRAE VPTAHPS PS P RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGT I GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKT PE PPVP CVPEQTEYAT IVFPSGMGT S SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO: 9)

Numbering of amino acid residues in PD-1 refers to the full length polypeptide shown in SEQ ID NO: 9. Amino acids 1-20 of SEQ ID NO: 9 define a signal sequence that is removed during translational processing resulting in the "mature PD1" molecule comprising amino acids 25-288 of SEQ ID NO: 9. Amino acids 168-190 of SEQ ID NO: 9 define the transmembrane domain and resides 191-288 define the cytoplasmic domain. The term PD-1 includes naturally occurring variants including the naturally occurring variant with the substitution of Alanine to Valine at position 215. Amino acids 25 -167 define the 150 amino acid extracellular domain of PD-1 having the amino acid sequence:

LDS PDR PWNPPT FS PA LLVVTEGDNA T FTCS FSNTS E S FVLNWYRM S PSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAI SLAP KAQI KE SLRA ELRVTERRAE VPTAHPSPSP RPAGQ FQ

(SEQ ID NO: 10)

The extracellular domain possesses four glycosylation sites at resides 49, 58, 74 and 116 and a disulfide bond exists between resides 54 and 123.

[0034] PD1 Receptor(s): As used herein, the term PD 1 receptor refers to either of the group consisting of B7-H1/PD-L1 (hereinafter "PD-L1") and B7-DC/PD-L2. hereinafter "PD- L2").

[0035] Polypeptide: The terms "polypeptide," "peptide," and "protein", used

interchangeably herein, refer to a polymeric form of amino acids of any length. A polypeptide may include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. Polypeptide includes but is not limited to fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N-terminus methionine residues; fusion proteins with immunologically tagged proteins.

[0036] It will be appreciated that throughout this disclosure reference is made to amino acids according to the single letter or three letter codes. For the reader's convenience, the single and three letter amino acid codes are provided in Table 1 below: Table 1. Amino Acid Abbreviations

G Glycine Gly

P Proline Pro

A Alanine Ala

V Valine Val

L Leucine Leu

I Isoleucine lie

M Methionine Met

C Cysteine Cys

F Phenylalanine Phe

Y Tyrosine Tyr

W Tryptophan Trp

H Histidine His

K Lysine Lys

R Arginine Arg

Q Glutamine Gin

N Asparagine Asn

E Glutamic Acid Glu

D Aspartic Acid Asp

S Serine Ser

T Threonine Thr

[0037] Prevent: The terms "prevent", "preventing", "prevention" are used

interchangeably to a refer to a course of action taken with respect to a subject to reduce the subject's risk of developing a disease, disorder, condition or delaying the onset thereof.

[0038] Recombinant: As used herein, the term recombinant to refer to polypeptides generated using recombinant DNA technology. The techniques and protocols for recombinant DNA technology are well known in the art.

[0039] Response: The term "response" refers to a change in biochemical or physiological behavior, e.g., concentration, density, adhesion, or migration within a biological compartment, rate of gene expression, or state of differentiation, where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms. In certain contexts, the terms

"activation", "stimulation", and the like refer to cell activation as regulated by internal mechanisms, as well as by external or environmental factors; whereas the terms "inhibition", "down-regulation" and the like refer to the opposite effects.

[0040] Specifically Binds: The term "specifically binds" as used herein refers to the situation in which one member of a specific binding pair does not significantly bind to molecules other than its specific binding partner(s) as measured by techniques well known in the including but not limited to competition ELISA, BIACORE® assays and/or KINEXA® assays. The term is also applied where the antigen-binding domain of an PBM of the present disclosure is specific for a particular epitope that may be present on multiple antigens, in which case the antigen- binding domain of the PBM specifically binds to the various antigens carrying the epitope. The term "epitope" refers to that portion of a molecule capable of being recognized by and bound by a PBM at one or more of the PBM's binding domains.

[0041] Substantially Pure: The term "substantially pure" is used to refer a sample wherein a component (e.g., a polypeptide) makes up greater than about 50% of the total content of the composition, and typically greater than about 60% of the total polypeptide content. When used in reference a solution, emulsion or suspension comprising a component, the contribution of the liquid phase is not calculated with respect to the calculation of percentage of total content of the composition. For example, a substantially pure aqueous solution of an agent (>95%) may consist primarily of water but the water is not factored in when calculating the percentage purity. More typically, "substantially pure" refers to compositions in which at least 75%, at least 85%, at least 90%) or more of the total composition is the component of interest. In some cases, the polypeptide will make up greater than about 90%, or greater than about 95% of the total content of the composition.

[0042] Subject/Patient: The terms "subject," and "patient" used interchangeably herein, refer to a mammal, such as a human suffering from a neoplastic condition. In a certain embodiment, a subject is further characterized with a disease or disorder or condition that would benefit from a decreased level of PD-1 activity. Examples of mammalian subjects include but are not limited to members of the superfamilies Cercopithecoidea and Hominoidea, in particular members of the family Hominidae including human beings. The term subject also includes members of the families Canidae (including Canis familiaris), Felidae (including Felinae and species of the genus Felis, in particular members of specifically including Felis catus), Equidae (specifically including species of the genus Equus such as domesticated horses), and Bovidae (including species of the tribe Bovini such as Bos taurus).

III. CDRs

[0043] The present disclosure provides polypeptide CDR sequences useful for the preparation of PDl-PBMs. The terms "complementary determining region" and its abbreviation "CDR" are those sequences typically provided in immunoglobulin molecules that determine the binding properties used herein to refer to the hypervariable domains of variable domains of immunoglobulins. Systematic identification of residues included in the CDRs have been developed by Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). The numbering of these CDR positions herein is provided according to Kabat numbering

conventions,

[0044] In particular, the present disclosure provides the CDRs useful in the preparation of PDl-PBMs as defined using the Kabat system in accordance with the present disclosure in Table 2 below:

IV. PDl-PBM

[0045] The present disclosure further provides a PD-1 polypeptide binding molecule

("PDl-PBM") comprising one or more polypeptide sequences selected from the group consisting of SEQ IDs#l-6. As used herein, the term PDl Polypeptide Binding Molecule ("PDl-PBM") refers to a polypeptide binding molecule comprising at least one or more of the CDRs SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 6, wherein the PDl-PBM specifically binds to the extracellular domain of a mammalian PDl and substantially inhibits the binding of the extracellular domain of a mammalian PDl at least one PDl receptor.

A. Polypeptide Binding Molecule

[0046] As used herein, the term Polypeptide Binding Molecule ("PBM") is used to refer collectively to the immunoglobulins (including but not limited to mammalian immunoglobulin classes IgGl, IgG2, IgG3 and IgG4) and immunoglobulin derivatives including but not limited to IgG(l-4)deltaCH2, F(ab')₂, Fab, ScFv, VH, VL, tetrabodies, triabodies, diabodies, dsFv, F(ab')₃, scFv-Fc and (scFv)₂.

[0047] PBMs of the present disclosure include monoclonal immunoglobulins (e.g., monoclonal antibodies). While a monoclonal immunoglobulin (e.g., monoclonal antibody) can be produced using hybridoma production technology, other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries).

1. Immunoglobulin

[0048] In one embodiment, the PDl-PBM is an immunoglobulin that specifically binds the extracellular domain of a mammalian PDl having an affinity for the extracellular domain of a mammalian PDl less than 10e-12 and substantially inhibits the binding extracellular domain of said mammalian PDl to bind to at least one PDl receptor.

[0049] The term "immunoglobulin" of "IgG" is used herein refers to a polypeptide consisting of subsequences substantially encoded by all or part of the recognized

immunoglobulin genes. For example, in humans, the immunoglobulin genes include the kappa ( ), lambda (λ), and heavy chain loci that comprise myriad variable region genes, and the constant region genes rau (υ), delta (δ), gamma (γ), sigma (σ), and alpha (a). The conventional mammalian immunoglobulin structure (e.g., human, as well as many other higher mammals) provides a tetrameric structure composed of two identical pairs of polypeptide chains, each pair having one "light" chain and one "heavy" chain. Light chain polypeptides (or "light chains") are classified as kappa and lambda light chains. Heavy chain polypeptides (or "heavy chains") are classified as rau, delta, gamma, alpha, or epsilon, and define the immunoglobulin's (e.g., antibody's) isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgGl , IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgMl and IgM2. IgA has several subclasses, including but not limited to IgAl and IgA2. By "isotype" as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgGl , IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE. The term IgG includes IgG fusions, where the IgG fusion contains sequences from two or more IgG molecules. The PDl -PBMs of the present disclosure may comprise sequences belonging to the IgG (including IgGl, IgG2, IgG3, IgG4 and fusions of any combination), IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG, IgM (and fusions of any combination) classes of antibodies, the IgG class being an embodiment of particular interest.

iM)5 | Each of the light and heavy chains of an immunoglobulin is comprised of two distinct regions, referred to in the art as the variable and constant regions. For example, the human IgG heavy chain is composed of four immunoglobulin domains linked from N- to C- terminus in the order VH-CH1-CH2-CH3, referring to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 respectively (also referred to as VH-Cy 1 -Cy2-Oy3 , referring to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain

respectively). Similarly, the human IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, referring to the light chain variable domain and the light chain constant domain respectively. The carboxy-terminal portion of each heavy chain defines a constant region primarily responsible for effector function.

[0051] By "variable region" as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including VK and νλ) and/or VH genes that make up the light chain (including kappa and lambda) and heavy chain immunoglobulin genetic loci respectively. The present disclosure provides variable region polypeptides (SEQ ID NO: 7 and SEQ ID NO: 8) useful in the preparation of PDl-PBMs of the present disclosure as provided in Table 3 below:

[0052] A light or heavy chain variable region (VL and VH) is composed of "framework"

(or "FR") regions interrupted by three hypervariable regions referred to as "complementarity determining regions" or "CDRs". There are 6 CDRs, three per each heavy and light chain, designated VH CDR1, VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDRS, respectively. Consistent with convention, the framework regions FR 1, FR2, FR3 and FR4 are separated by the CDRs (CDR 1 , CDR2 and CDR3). The extent of the framework region and CDRs have been precisely defined, for example as in Kabat (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, (1983)). The framework regions of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen and provides a stable scaffold upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens.

2. Immunoglobulin Derivatives:

[0053] The term "immunoglobulin derivative" refers to a PBM derived from an immunoglobulin. The term "derived from" in the context of a PBM derived from an

immunoglobulin refers to both physical isolation of elements from an immunoglobulin as well as sequence information derived from an isolated immunoglobulin. Consequently, for example, an immunoglobulin derivative may be wholly synthetic but based on sequence information derived from an isolated naturally occurring or engineered immunoglobulin and be considered "derived from" the immunoglobulin.

[0054] Immunoglobulin derivatives include antigen-binding fragments of an antibody, as well as polypeptides having the antigen-binding components of an antibody (e.g., single chain antibodies, where the heavy and light chains of the antibody or joined via a linker). Examples of immunoglobulin derivatives include IgG(l-4)deltaCH2, F(ab')₂, Fab, ScFv, VH, VL, tetrabodies, triabodies, diabodies, dsFv, F(ab')₃, scFv-Fc and (scFv)₂ molecules. The term F(atv)2 refers to a bivalent PBM comprising two linked Fab fragments. The term Fab refers to a PBM consisting of VL, VH, CL and CHI domains of an immunoglobulin. The term Fv refers to a PBM consisting of the VL and VH domains of a single immunoglobulin parent. The term ScFv refers to a single chain pBM comprising the light and heavy chain variable regions (LCVR and HC VR) with a linker sequence. (See, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol . 1 13, Rosenburg and Moore eds., Springer- Verlag, New York, pp 269-315, 1994). ScFv molecules may be joined to a CH3 domain to create scFv-Fc molecules also referred to in the art as "minibodies,"

[0055] The term VH refers to a polypeptide chain PBM corresponding substantially to the heavy chain variable region of the parent antibody. The term VL refers to a polypeptide chain PBM corresponding substantially to the light chain variable region of the parent antibody.

Multiple VH and/or VL polypeptides may be joined by linker peptides to create multivalent compounds commonly referred to diabodies (bivalent), triabodies (trivalent) and tetrabodies (quadravalent).

[0056] The foregoing immunoglobulins and immunoglobulin derivatives may be derived from germline sequences (i.e. set of sequences that compose the natural genetic repertoire of antibodies and their associated alleles) or may synthetic by combining various domains from different immunoglobulin molecules to engineer a PBM having specific combinations of properties. For example, the CDR polypeptides of the present disclosure provided may be incorporated into any of a variety of framework regions to define synthetic VL and VH sequences.

[0057] In one embodiment the PDl-PBM is a human, humanized or deimmimized immunoglobulin molecule comprising one or more of the CDRs of SEQ ID NOS: 1, 2, 3, 4, 5, and 6. In one embodiment, the PDl-PBM is a human, humanized or deimmimized

immunoglobulin molecule (e.g., antibody, antigen-binding fragment of an antibody) having a VH having the CDRs of the heavy chain of SEQ ID NO: 8 (VH-09), a VL having the CDRs of the light chain of SEQ ID NO:7 (VL-09), or both.

[0058] In one embodiment, the PDl-PBM is a human, humanized or deimmunized immunoglobulin molecule (e.g., antibody, antigen-binding fragment of an antibody) having a VH having CDRs of amino acid sequences of SEQ ID NO:4 (CDR-Hl ), SEQ ID NO: 5 (C DREE.) and SEQ ID NO:6 (CDR-H3). In one embodiment, the PDl-PBM is a human, humanized or deimmunized immunoglobulin molecule (e.g., antibody, antigen-binding fragment of an antibody) having a VL having CDRs of amino acid sequences of SEQ ID NO: l (CDR-Ll), SEQ ID NO:2 (CDR-L2) and SEQ ID NO: (CDR-L3). In one embodiment, the PDl-PBM is a human, humanized or deimmunized immunoglobulin molecule (e.g., antibody, antigen-binding fragment of an antibody) having a VH having CDRs of amino acid sequences of SEQ ID NO:4 (CDR-Hl), SEQ ID NO: 5 (CDR-H2) and SEQ ID NO:6 (CDR-H3) and a VL having CDRs of amino acid sequences of SEQ ID NO: 1 (CDR-Ll), SEQ ID NO 2 (CDR-L2) and SEQ ID NO: 3 (CDR-L3). [0059] The term "humanized" immunoglobulin is used herein to refer to an immunoglobulin comprising portions of immunoglobulins of different origin, wherein at least one portion comprises amino acid sequences of human origin and at least one portion is from a non-human origin (e.g., artificial origin (e.g., non-naturally occurring) or non-human origin). In conventional nomenclature, the immunoglobulin providing the CDR's is called the "donor" and the immunoglobulin providing the framework is called the "acceptor".

[0060] The term "deimmunized immunoglobulin" refers to immunoglobulin which has one or more heavy chain framework regions, light chain framework regions, light chain constant regions, and/or heavy chain constant regions modified to reduce immunogenicity of the immunglobulin, e.g., to remove T cell epitopes that may induce an anti-immunoglobulin immune response (e.g., following administration to a human subject).

[0061] In some embodiments, the PD1-PBM of the present disclosure incorporates the Fc region of an immunoglobulin. The term "Fc" or "Fc region", as used herein is meant the polypeptide comprising the constant region of an immunoglobulin excluding the first constant region immunoglobulin domain. Thus Fc refers to the C-terminal constant region

immunoglobulin domains of IgA, IgD, and IgG, and the three C-terminal constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. With respect to IgA and IgM, the Fc region may include the J chain. With respect to IgG, the Fc region comprises immunoglobulin domains Cgamnia2 and Cgamma3 (Cy2 and Oy3) and the hinge between Cgammal (Oyl) and Cgamma2 (Cy2). Although the boundaries of the Fc region may vary in certain contexts, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus.

[0062] In one embodiment, an PD1-PBM is a mammalian immunoglobulin or "full length antibody" or "antibody" comprising variable and constant domains providing binding and effector functions. In most instances, a full length antibody comprises two light chains and two heavy chains, each light chain comprising a variable region and a constant region. Constant regions may be derived from one or more IgG subclasses including IgGl, IgG2, IgG3 and igG4. In one embodiment, the constant and variable regions are "human" (i.e. possessing amino acid sequences characteristic of human immunoglobulins)

[0063] The present disclosure provides PDl-PBMs that specifically bind the extracellular domain of mature PD1 (SEQ ID NO: 10).

[0064] The present disclosure also provides PDl-PBMs (e.g., antibodies, antigen-binding antibody fragments) that specifically bind an epitope of PD-1, wherein the PD1-PBM competes for binding with an antibody that comprises light chain CDRs of an antibody light chain variable region comprising amino acid sequence SEQ ID NO: 7 and heavy chain CDRs of an antibody heavy chain variable region comprising amino acid sequence SEQ ID NO:8. The present disclosure also provides PDl-PBMs that specifically bind an epitope of PD-1, wherein the PD1- PBM competes for binding with an antibody having i) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of DISGGGGTTYYADSVKG (SEQ ID NO:5), and a CDR3 comprising the amino acid sequence of SGTVVTDFDY (SEQ ID NO:6), and ii) a light chain variable region comprising a CDR1 comprising the amino acid sequence of

GGNSIGSYSVH (SEQ ID NO: 1), a CDR2 comprising the amino acid sequence of DDSDRPS (SEQ ID NO: 2), and a CDR3 comprising the amino acid sequence of QVWDTSSYWV (SEQ ID NO: 3).

[0065] As used herein, a PD1-PBM "competes for binding" with a reference anti-PD-1 antibody where specific binding of the PD1-PBM to PD-1 inhibits binding of the reference anti- PD-1 antibody to PD-1 or vice versa. PDl-PBMs compete for binding with a reference anti-PD-1 antibody when, for example, the PD1-PBM binds the same or overlapping epitope bound by the reference anti-PD-1 antibody. Competitive binding assays can be conducted under physiological conditions to detect specific binding to an extracellular region of PD-1, e.g., as presented on a surface of a PD-1 expressing cell, using conventional methods. PDl-PBMs that compete for binding with a reference anti-PD-1 antibody can provide for an activity of the reference anti-PD- 1 antibody, e.g., inhibition of binding of one or both of PD-L1 (B7-H1) and PD-L2 (B7-DC) to cell-surface PD-1. [0066] The present disclosure also provides PDl-PBMs (e.g., antibodies, antigen-binding antibody fragments) that specifically bind an epitope of PD-1 bound by with an antibody that comprises light chain CDRs of an antibody light chain variable region comprising amino acid sequence SEQ ID NO:7 and heavy chain CDRs of an antibody heavy chain variable region comprising amino acid sequence SEQ ID NO:8. The present disclosure also provides PD l- PBMs that specifically bind an epitope of PD-1 bound by an antibody having i) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of DISGGGGTTYYADSVKG (SEQ ID NO:5), and a CDR3 comprising the amino acid sequence of SGTVVTDFDY (SEQ ID NO:6), and ii) a light chain variable region comprising a CDR1 comprising the amino acid sequence of GGNSIGSYSVH (SEQ ID NO: 1), a CDR2 comprising the amino acid sequence of DDSDRPS (SEQ ID NO: 2), and a CDR3 comprising the amino acid sequence of

QVWDTSSYWV (SEQ ID NO: 3).

[0067] In one embodiment of the disclosure, the PD1-PBM is AM0001 : a monoclonal antibody with a lambda 2 light chain and an IgG4 with a serine to proline substitution at position 228 (S228P) to provide a "hinge-stabilized" heavy chain, characterized by VL and VH CDRs having amino acid sequences corresponding to SEQ ID NOS: 1- 6 as set out in Table 2 hereinabove, a light chain variable region characterized by the sequence of SEQ ID NO: 7 and a heavy chain variable region characterized by the amino acid sequence of SEQ ID NO:8. The AM0001 antibody is characterized as having a binding affinity (Kd) for human and

cynomologous monkey PD-1 of about 10 pM or less at 25° C (as determined by biolayer micrometry). AMOOO l may be prepared in substantial accordance with the teaching of Example 1 herein. The binding affinity of M9 and AMOOOl, measured by bio-layer interferometry (BLI, Octet Red 96, Forte Bio, Menlo Park, CA), are shown in Table 4 below.

Table 4. PD-1 Binding Affinities of M9 and AMOOOl

Antibody KD(M) Kon Kdis R²

M9 7.023E-1 1 6.448E+5 4.529E-5 0.9995

AMOOOl < 1.0E-12 6.655E+5 <1.0E-7 0.9989 [0068] The full length amino acid sequences of the heavy chain and light chain of

AMOOOl are provided below:

AMOOOl Mature Heavy Chain Protein Sequence (Human IgG4 S228P Framework):

EVQLLESGGGLVQPGGSLRLSCPASGFTFSSYAMSWVRQAPGKGLGWVSDISGG

GGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCAKSGTVVTDFD

YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS

GALTSGVHTFP AVLQ S SGLYSLS S VVTVP S S SLGTKT YTCNVDHKP SNTK VDKRV

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ

FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK

GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE

SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY

TQKSLSLSLGK (SEQ ID NO: 11)

AMOOOl Mature Light Chain Protein Sequence (Human Lambda-2 Framework):

SYVLTQPPSVSVAPGQTARVTCGGNSIGSYSVHWYQQKPGQAPVLVVYDDSDRP SGIPERFSGSNSGNTAALTISRVEAGDEADYYCQVWDTSSYWVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 12)

3. Conjugates:

[0069] The PD1 -PBMs of the present disclosure may be conjugated or operably linked to another therapeutic compound. The therapeutic compound may be a cytotoxic agent, a chemotherapeutic agent, a toxin, a radioisotope, a cytokine, or other therapeutically active agent.

[0070] The PDl-PBMs of the present disclosure may be conjugated to a protein or molecule for utilization in tumor pretargeting or prodrug therapy. [0071] The PDl-PBMs of the present disclosure may be linked to one of a variety of non-proteinaceous polymers, for example e.g., polyethylene glycol (PEG).

Diagnostic:

[0072] The present disclosure further provides a method for detecting PD-1 protein in a biological sample said method comprising contacting incubating a PDl-PBM of the disclosure with the biological sample under conditions and for a time sufficient to permit said PDl -PBM to bind to PD-1 protein, and detecting said binding.

Imaging Agent:

[0073] The present disclosure further provides an imaging agent said imaging agent comprising a PD l-PBM molecule in stable association (e.g. covalent, coordinate covaient) with an imaging agent. The term imaging agent is used to describe any of a variety of compounds a signature that facilitates identification, tracing and/or localization of the PDl-PBM (or its metabolites) using diagnostic procedures. Examples of imaging agents include but are not limited to fluorescent compounds, radioactive compounds, compounds opaque to imaging methods (e.g. X-ray, ultrasound).

V. Production of PDl-PBMs

[0074] The PDl-PBMs of the present disclosure may be prepared using conventional techniques well known to those of skill in the art. Weil established methods for antibody molecular biology, expression, purification, and screening are described in Antibody

Engineering, edited by Duebel & Kontermann, Springer- Verlag, Heidelberg, 2001 ; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Anna Rev Biomed Eng 2:339-76; Antibodies: A Laboratory Manual by Harlow & Lane, New York: Cold Spring Harbor Laboratory Press, 1988.

[0075] The present disclosure further provides isolated nucleic acid molecules encoding the VH, VL, heavy chain and/or light chain of an antibody of the present disclosure: a vector comprising that nucleic acid, optionally operabiy-linked to control sequences recognized by a host ceil transformed with the vector; a host cell comprising that vector; a process for producing an antibody of the present disclosure comprising cuituring the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture medium. A polynucleotide is "operably linked" when it is placed into a functional relationship with another polynucleotide. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. The heavy and light chains of the antibody can be provided on the same or different vectors, and may be expressed from the same or different promoters.

[0076] For example, in one embodiment wherein the PD1-PBM is an immunoglobulin or derivative ("PDl-PBMabs"), nucleic acids are created that encode the PD1 -PBMabs and that may then be cloned into host cells, expressed and assayed, if desired. DNA is synthesized in accordance with well-known procedures. For example, a variety of methods that may find use in generating PDl-PBMabs herein are described in Molecular Cloning— A Laboratory Manual, 3^ld Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001. Additionally, there are a variety of commercially available kits and methods for gene assembly, mutagenesis, vector subcloning, and the like, and useful in generating nucleic acids that encode PDl-PBMabs.

[0077] The present disclosure provides recombinant host cells (e.g., yeast cells, mammalian cells) containing a nucleic acid comprising a nucleotide sequence encoding a variable heavy chain polypeptide and a nucleic acid comprising a nucleotide sequence encoding a variable light chain polypeptide, wherein the variable heavy and light chains have the CDRS, or the full-length sequence, of a variable heavy and light chain polypeptides of a D l -PBM as disclosed herein. The nucleic acids encoding the heavy and light chains of a PD1-PBM may be on the same or different expression constructs in the host ceil, and may optionally be integrated into the host ceil genome. In one embodiment, the variable heavy chain comprises a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of DISGGGGTTYYADSVKG (SEQ ID NO:5), and a CDR3 comprising the amino acid sequence of SGTWTDFDY (SEQ ID NO:6). In one embodiment, the variable light chain comprises a CDR1 comprising the amino acid sequence of

GGNSIGSYSVH (SEQ ID NO: 1), a CDR2 comprising the amino acid sequence of DDSDRPS (SEQ ID NO: 2), and a CD 3 comprising the amino acid sequence of QVWDTSSYWV (SEQ ID NO: 3). In one embodiment, the variable heavy chain comprises the amino acid sequence of SEQ ID NO: 8. In one embodiment the variable light chain comprises the amino acid sequence of SEQ ID NO: 7.

[0078] The present disclosure provides expression vectors having a nucleic acid comprising a nucleotide sequence encoding a variable heavy chain polypeptide, and/or a nucleic acid comprising a nucleotide sequence encoding a variable light chain, wherein the variable heavy and light chains have the CDRS, or the full-lengt sequence, of a variable heavy and light chain polypeptides of a PDl-PBM as disclosed herein. The nucleic acids encoding the heavy and light chains of a PDl-PBM may be on the same or different expression vectors, and the nucleic acids encoding the variable heavy and variable light chains may be operably linked to the same or different promoters. In one embodiment, the variable heavy chain comprises a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of DIS GGGGTTYYAD S VKG (SEQ ID NO: 5), and a CDRS comprising the amino acid sequence of SGTVVTDFDY (SEQ ID NO: 6). In one embodiment, the variable light chain comprises a CDR1 comprising the amino acid sequence of

GGNSIGSYSVH (SEQ ID NO: 1), a CDR2 comprising the amino acid sequence of DDSDRPS (SEQ ID NO: 2), and a CDR3 comprising the amino acid sequence of QVWDTSSYWV (SEQ ID NO: 3). In one embodiment the variable heavy chain comprises the amino acid sequence of SEQ ID NO: 8. In one embodiment the variable light chain comprises the amino acid sequence of SEQ ID NO: 7. The disclosure further provides a recombinant host cell comprising an expression vector(s) as disclosed herein.

[0079] The nucleic acid sequence encoding the PDl-PBMs are typically incorporated into a vector for expression which are then used to transform a host cell to effect expression of the protein in the host cell. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been operably linked including, but not limited to, plasmids and viral vectors. Certain vectors are capable of autonomous replication in a host ceil into which they are introduced while other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby, are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Exemplary vectors are well known in the art.

[0080] The nature of the vector depends on the nature of the host cell used for expression. As used herein, the expressions "cell," "host cell," and "cell culture" are used interchangeably and include an individual cell or cell culture that is a recipient of any isolated polynucleotide of the present disclosure or any recombinant vector(s) comprising a nucleotide sequence encoding a HCVR, LCVR or antibody of the present disclosure. A host cell includes ceils genetically modified (e.g., transformed, transduced or infected) with a polynucleotide (e.g., in one or more recombinant vectors) for expression of a PD1-PB of the present disclosure or a light chain or heavy chain thereof. For example, the PDl-PBMs may be expressed in a mammalian expression system, including systems in which the expression constructs are introduced into the mammalian cells using virus. A variety of mammalian cells may be used for such recombinant expression including but not limited to NIH3T3, CHO, COS, HEK293, PER C.6 and HeLa cells. In an alternate embodiment, PDl -PBMs may be expressed in bacterial ceils. Bacterial expression systems are well known in the art, and include Escherichia coli (E, coli. Alternatively, the PDl -PBMs may be produced in insect cells (e.g. Sf21 /Sf9, Trichoplusia ni Bti- Tn5bl-4) or yeast cells (e.g. S. cerevisiae, Pichia, etc). In addition, the PDl-PBMs may be produced by chemical synthesis methods. Also transgenic expression systems both animal (e.g. cow, sheep or goat milk, embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn, tobacco, duckweed, etc.). The conditions appropriate for expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.

[0081] In order to facilitate expression, purification, screening, etc. the PDl -PBMs may be operably linked to a tag sequence joined via a linker sequence. Typically, the linker sequence will comprise a small number of amino acids, typically less than ten, and are selected to be conformationally flexible and resistant to degradation such as GGGGS. A tag sequence may be a targeting or signal sequence that directs the recombinantly express PDl-PBMs fusion protein to a desired cellular location or to the extracellular media. The tag may also be a sequence that that facilitates purification and/or screening such as polyhistidme tags (His-tags), GST fusions, MBP fusions and epitope tags such as myc tags, flag-tags, and the like. In the alternative to direction expression of a PDI-l-PBM fusion protein, a tag may be covalent!y conjugated to the PD1 -PBM by traditional chemical means,

[0082] In one embodiment, the recombinantly expressed PDl-PBMs are purified or isolated after expression using conventional chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed~pha.se, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC.

Purification may also be achieved by methods also include electrophoretic, immunological, dialysis, and filtration techniques, in conjunction with protein concentration, are also useful.

[0083] In one embodiment of the present disclosure, when the PD1-PBM is AM000I , the nucleic acid sequences encoding the heavy and light chains are as set out below:

AMOOOl Mature Heavy Chain DNA Sequence (Human IgG4 S228P Framework):

GAGGTCCAGCTCCTGGAATCCGGGGGCGGTCTGGTCCAGCCGGGCGGCTCGCTCCG

CCTGTCCTGCCCGGCGAGCGGCTI ::ACCTTCTCCTCCTA(XJCCATGT

GCAGGCCCCCGGCAAGGGCCTCGGCTGGGTCAGCGACATCTCCGGCGGCGGCGGCA

CCACGTACTACGCGGACTCGGTGAAGGGCCGGTTCACGATCTCCCGGGACAACTC^

AAGAACACCCTGTACCTGCAGATGAACTCACTGCGGGGCGAGGACACGGCGGTGTA

TTACTGCGCCAAGTCCGGAACGGTTGTGACTGATTTCGACTACTGGGGCCAGGGCAC

CCTGGTGACCGTGTCCAGCGCCTCCACCAAGGGCCCCAGCGTGTTCCCCCTGGCGCC

GTGCTCGCGGAGCACCAGCGAGTCCACCGCCGCGCTCGGTTGCCTCGTCAAGGACT

ACTTCCCCGAGCCGGTCACAGTGTCATGGAACTCCGGCGCGCTGACGAGCGGCGTG

CACACCTTCCCGGCCGTGCTCCAGTCCAGCGGCCTGTACAGCCTCAGTAGCGTCGTG

ACGGTGCCCTCGTCGTCGCTGGGCACGAAGACCTACACCTGCAACGTGGAC^

GCCGTCCAACACCAAGGTCGATAAGCGAGTGGAGAGCAAGTACGGCCCCCCGTGCC CCCCCTGCCCGGCCCCGGAGTTCCTGGGTGGCCCCTCCGTGTTCCTCTTCCCCCCGAA

GCCCAAAGACACCCTCATGATCAGCCGGACGCCGGAGGTCACGTGCGTCGTCGTGG

ACGTGAGCCAGGAAGACCCGGAGGTCCAGTTCAACTGGTACGTGGACGGCGTCGAG

GTGCATAACGCCAAGACCAAGCCTCGCGAGGAACAGTTCAACTCCACTTACCGCGT

CGTGTCCGTCCTCACCGTCCTGCACCAGGACTGGCTCAACGGGAAGGAATACAAGT

GCAAGGTCTCGAACAAGGGCCTGCCGTCGTCCATCGAGAAGACCATCAGCAAGGCC

AAGGGCCAGCCGCGGGAGCCCCAGGTCTACACCCTCCCCCCCTCCCAGGAAGAGAT

GACGAAGAACCAGGTGAGCCTGACGTGCCTCGTGAAGGGGTTCTACCCCTCCGACA

TCGCAGTCGAGTGGGAGAGCAACGGCCAGCCGGAGAACAACTACAAGACGACCCC

CCCGGTGCTGGACAGCGACGGGTCCTTCTTCCTCTACTCGCGTCTCACAGTCGACAA

GTCGCGCTGGCAGGAGGGCAACGTCTTCTCGTGCTCCGTGATGCACGAGGCCCT^

CAACCACTACACCCAGAAGTCGCTGTCCCTGTCCCTGGGCAAG (SEQ ID NO: 13)

AMOOOl Mature Light Chain Protein Sequence (Human Lambda-2 Framework):

AGCTACGTGCTGACCCAGCCGCCCTCGGTGTCGGTCGCCCCGGGCCAGACGGCACG

TGTGACCTGCGGCGGTAACAGCATCGGCTCCTACTCGGTCCACTGGTATCAGCAGAA

GCCGGGGCAGGCCCCGGTCCTGGTGGTCTACGACGACAGCGACCGCCCGTCCGGCA

TCCCCGAACGCTTCAGCGGCTCAAACAGCGGGAACACCGCGGCCCTGACGATCTCG

CGCGTCGAGGCGGGGGACGAAGCCGATTACTACTGCCAGGTCTGGGACACCTCGAG

TTACTGGGTGTTCGGCGGGGGCACGAAGCTGACCGTCCTCGGCCAGCCGAAGGCCG

CCCCCTCAGTAACCCTGTTCCCCCCGTCCTCGGAGGAGTTGCAGGCGAACAAGGCGA

CGCTGGTGTGCTTGATCTCGGACTTCTACCCCGGAGCGGTGACGGTCGCCTGGAAGG

CCGACTCCTCCCCGGTCAAGGCGGGCGTGGAGACGACCACCCCCTCCAAGCAGAGC

AACAACAAGTACGCCGCCTCGAGCTACCTCTCGCTGACACCCGAGCAGTGGAAGTC

CCACCGGTCCTACTCGTGCCAGGTAACCCACGAGGGCTCCACCGTCGAGAAGACCG

TGGCCCCCACCGAGTGCAGC (SEQ ID NO: 14) VI. Pharmaceutical Formulations

[0084] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an PDl-PBM of the present disclosure and a pharmaceutically acceptable carrier or diluent. In one embodiment, the pharmaceutical composition comprises a homogeneous or substantially homogeneous population of a PDl-PBM of the present disclosure and a pharmaceutically acceptable carrier or diluent. The present disclosure further provides the use of a PDl-PBM of the present disclosure for use in the preparation of a medicament.

[0085] A PD l-PBM of the present disclosure may be provided in a pharmaceutical formulation suitable for administration to a subject. A PDl-PBM of the present disclosure may be administered to a subject directly but is more commonly provided in a pharmaceutical formulation comprising one or more pharmaceutically acceptable diluents, carriers, and/or excipients such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonic! ty agents, and/or stabilizing agents. Said compositions can be designed in accordance with conventional techniques disclosed in, e.g., Biological Drug Products: Development and Strategies (201.3) Wang and Singh. Eds. 2013, Wiley; Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic (2012) Meyer, Ed. Woodhead Publishing; and Remington, The Science and Practice of Pharmacy, 22^nd Edition, (2012) Loyd, et al, lids. Pharmaceutical Press, which provide formulation techniques generally available to those of skill in the art.

[0086] In order to provide enhanced stability of protein products, the formulation may be provided in a lyophilized powder form. In such presentation the formulation is typically provided in a kit of parts comprising vial containing the lyophilized formulation of the PDl- PBM and a quantity of reconstitution buffer. Alternatively, the formulation may be provided in a dual chamber pre-filled deliver}' device which facilitates reconstitution of the lyophilized PDl- PBM formulation with a buffer solution and delivery by either intravenous, intratumoral, intradermal, intraperitoneal, intracranial, intraocular, subcutaneous, and/or intramuscular routes of parenteral administration. VII. Methods of Treatment:

[0087] The present disclosure provides a method of treating or preventing a neoplastic disease in a subject by the administration of a therapeutically effective amount of a PDl-PBM. The compositions of the present disclosure are useful in the treatment of mammalian subjects.

[0088] The terms "treat", "treating", treatment" are used interchangeably to refer to the administration of a compound of the present disclosure for treatment of a disease or condition in a subject to a subject suffering from a disease, disorder or condition, or a symptom thereof to temporarily or permanently eliminate, reduce, suppress, mitigate, or ameliorate a cause or symptom of such disease, disorder, or condition but does not necessarily indicate a total elimination of all disorder symptoms. The term treatment includes the suspension or slowing the progression of the disease, disorder or condition or inhibiting progression thereof to a harmful or otherwise undesired state.

[0089] As used herein, the term "neoplastic disease" refers to disorders or conditions in a subject arising from cellular hyper-proliferation or unregulated (or dysregulated) cell replication. The term neoplastic disease refers to disorders arising from the presence of neoplasms in the subject. Neoplasms may be classified as: (1) benign (2) pre-malignant (or "pre-cancerous"); and (3) malignant (or "cancerous"). Examples benign neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to adenomas, fibromas, hemangiomas, and lipomas. Examples of pre-malignant neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to hyperplasia, atypia, metaplasia, and dysplasia. Examples of malignant neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to carcinomas (cancers arising from epithelia tissues such as the skin or tissues that line internal organs), leukemias, lymphomas, and sarcomas typically derived from from bone fat, muscle, blood vessels or connective tissues). Examples of neoplastic diseases amenable to treatment with the compositions and methods of the present disclosure include breast cancers; sarcomas (including but not limited to osteosarcomas and angiosarcomas) , and fibrosarcomas), leukemias, lymphomas, genitourinary cancers (including but not limited to ovarian, urethral, bladder, and prostate cancers); gastrointestinal cancers (including but not limited to colon esophageal and stomach cancers); lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders; cervical carcinoma-in-situ; intestinal polyposes; oral leukoplakias; histiocytoses, hyperprofroliferative scars including keloid scars, hemangiomas; hyperproliferative arterial stenosis, psoriasis, inflammatory arthritis;

hyperkeratoses and papulosquamous eruptions including arthritis. Also included are viral induced neoplasms such as warts and EBV induced disease (i.e., infectious mononucleosis), scar formation, hyperproliferative vascular disease including intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion and the like.

[0090] The terms "administration", "administer" refer to contact of a subject, cell, tissue, organ, or biological fluid with a PD1-PBM, e.g., a pharmaceutical formulation comprising a PD1-PBM. In the context of a cell, administration includes contact (in vitro or ex vivo) of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. In one embodiment of the present disclosure, the compositions of the present disclosure are administered by intravenous infusion.

[0091] The phrase "therapeutically effective amount" refers to the quantity of a therapeutic or prophylactic agent that when administered to a subject suffering from a disease, disorder of condition that provides a detectable, positive objective effect or subjective positive effect (e.g., a subject's feeling of well-being) on any symptom, aspect, or characteristic of the disease, disorder or condition. A therapeutically effective amount can be ascertained by measuring observable physiological parameters (e.g., body temperature, blood pressure, etc.) or through diagnostic analysis of the subject (e.g. x-ray, ultrasound, CT-scan, analysis of bodily fluids).

[0092] The determination of a therapeutically effective amount of the compositions of the present disclosure is capable to routine determination by the skilled physician based on readily identifiable indicia such as age, weight, sex, general health, blood pressure, additional conditions (e.g. diabetes, metabolic syndrome), disease type, disease progression, tumor burden as well as the therapeutic modality such as whether the compositions are being administered in combination with other the co-administration of other agent which may lead to incompatibilities or cross-reactions.

[0093] In one embodiment, of compositions of the present disclosure a therapeutically effective amount in the treatment of a neoplastic disease is a dose of approximately 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg or 50 mg/kg provided daily for a period of 5 days by intravenous infusion.

VIII. Combination with Additional Therapeutic Agents

[0094] The compositions of the present disclosure may be administered in combination with additional anti -neoplastic therapeutic agents or therapies. Examples of such additional antineoplastic therapeutic agents include immune checkpoint inhibitors, immunomodulatory compounds, chemotherapeutic agents and physical methods.

A. Checkpoint Inhibitors:

[0095] Examples of immune checkpoint inhibitors include PD1 antagonists (e.g. anti-

PD1 antibodies), PDL1 antagonists (e.g. anti-PDLl antibodies), PDL2 antagonists (e.g. anti- PDL2 antibodies), CTLA-4 antagonists (e.g. anti-CTLA-4 antibodies), IDO inhibitors, 2B4 (also known as CD244) receptor antagonists, B7 family inhibitory !igands such B7-H3 (also known as CD276), B7-H4 (also known as B7-S1, B7x and VCTN1), and LAG3 antagonists (e.g. anti- LAG3 antibodies). Exemplary anti-PDl antibodies useful for use in combination with the compositions of the present disclosure include but are not limited to nivolumab (Bristol-Myers Squibb), and lambrolizumab (Merck)). Exemplary anti -CTLA-4 antibodies useful for use in combination with the compositions of the present disclosure include but are not limited to ipilimumab. Exemplary anti-PDLl antibodies useful for use in combination with the

compositions of the present disclosure include but are not limited to BMS-936559 (Bristol- Myers Squibb), MPDL3280A (Genentech/Roche) and MEDI4736 (Medlmmune). Exemplary anti-PDL2 antibodies useful for use in combination with the compositions of the present disclosure include but are not limited to AMP -224 (Amplimmune/GlaxoSmithKline)). Examples of a LAG-3 antagonists includes but is not limited to ΓΜΡ321 (ImmuFact). They include both receptors (e.g., the 2B4 (also known as CD244) receptor) and ligands (e.g., certain B7 family inhibitory ligands such B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7x and VCTN1 )). B7-H3, a !igand that inhibits T-cell activation (MGA271 ; MacroGenics) is another example of a checkpoint inhibitor. Examples of IDO inhibitors include Indoximod (NewLink Genetics).

B- Immun om odu 1 atory Agents :

[0096] Exemplary irn.munomodulat.ory agents useful for use in combination with the compositions of the present disclosure include but are not limited to tumor vaccines, IL-10, IL- 12, pegyiated IL-10, TIM3 antagonists (e.g. anti-TIM3 antibodies), gaiectin 9 antagonists, adenosine A2a receptor A2aR (A2a.R) antagonists that block binding by adenosine or by adenosine analogs. See, e.g., Pardoli, (April 2012) Nature Rev. Cancer 12:252-64; Zhu et al, (2005) Nature Immunol, 6: 1245-52, Ngiow et al, (201 1) Cancer Res. 71 :3540-51).

1. IL-10

[0097] IL-10 is an example of an immumodulatory agent particularly useful in combination with the PD1 -PBMs of the present disclosure. The terms "IL-10", "IL-10 polypeptide(s), "IL-10 molecule(s)", "IL-10 agent(s)" are used interchangeably herein to refer to an isolated naturally occurring form of an IL-10 molecule, with or without its signal peptide, including analogs, variants, muteins and active fragments thereof. Naturally occurring mammalian forms of IL-10 are well known in the art and include but are not limited to human IL-10 ("hIL-10", NP_000563), murine IL-10 ("mIL-10", NP_034678), rat IL-10 ("rIL-10", accession NP_036986.2; GI 148747382); bovine IL-10 ("bIL-10", accession NP_776513.1; GI 41386772); equine IL-10 ("eIL-10", accession NP_001009327.1; GI 57164347); canine IL-10 ("cIL-10", accession ABY86619.1; GI 166244598); and rabbit IL-10 ("rbIL-10", accession AAC23839.1; GI 3242896). Naturally occurring IL-10 species are biologically active (interact with and activate the IL-10 receptor) as a homodimer each subunit containing a mature IL-10 polypeptide and a signal peptide. Human IL-10 in its biologically active form exists as a homodimer with a molecular mass of 37kDa, comprising two 18.5kDa monomers (see, e.g., US Patent No. 6,217,857). Each hIL-10 monomer is expressed as a single polypeptide sequence of 178 amino acids, the N-terminal 18 residues comprising a signal peptide which is cleaved during post-translational processing to yield a mature 160 amino acid monomer. Each monomer contains two cysteine residues that form an intra-chain disulfide bond. Two mature IL-10 monomers associate non-covalently to form the fully active IL-10 dimer. When the IL-10 polypeptide is produced by recombinant DNA technology, the abbreviation may be preceded by an "r" to denote recombinantly produced as in "rhIL-10" referring to a human IL-10 peptide produced by recombinant DNA technology.

[0098] The term "active fragments" refers to polypeptide sequences containing contiguous amino acid residues derived from the mature IL-10 that retain IL-10 activity. The length of contiguous amino acid residues of a peptide or a polypeptide subsequence varies depending on the specific naturally-occurring amino acid sequence from which the subsequence is derived. In general, active fragments can be from about 20 amino acids to about 40 amino acids, from about 40 amino acids to about 60 amino acids, from about 60 amino acids to about 80 amino acids, from about 80 amino acids to about 100 amino acids, from about 100 amino acids to about 120 amino acids, from about 120 amino acids to about 140 amino acids, from about 140 amino acids to about 150 amino acids, from about 150 amino acids to about 155 amino acids, from about 155 amino acids up to the full-length peptide or polypeptides.

[0099] The term "IL-10 analogs" refers to polypeptides comprising an amino acid sequence having at least 75%, at least 80%>, at least 85%>, at least 90%, at least 95%, at least 98%>, or at least 99%, amino acid sequence identity to a contiguous stretch of at least 20 amino acids, 40 amino acids, 60 amino acids, 80 amino acids, 100 amino acids, 120 amino acids, 140 amino acids, 150 amino acids, 155 amino acids, from about 155 amino acids up to the full-length an IL- 10 polypeptide and exhibiting at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the activity of the IL-10 polypeptide Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[00100] The term IL-10 includes IL-10 variants. As used herein, the term "variant" encompasses naturally-occurring variants and non-naturally-occurring variants. Naturally- occurring variants include homologs (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, from one species to another), and allelic variants

(polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, from one individual to another within a species). Non-naturally-occurring variants include polypeptides and nucleic acids that comprise a change in amino acid or nucleotide sequence, respectively, where the change in sequence is artificially introduced (e.g., muteins); for example, the change is generated in the laboratory by human intervention ("hand of man"). Thus, herein a "mutein" refers broadly to mutated recombinant proteins that usually carry single or multiple amino acid substitutions and are frequently derived from cloned genes that have been subjected to site-directed or random mutagenesis, or from completely synthetic genes.

[00101] The term IL-10 variants include IL-10 molecules comprising conservative amino acid substitutions. The phrase "conservative amino acid substitution" refers to substitutions that preserve the activity of the protein by replacing an amino acid(s) in the protein with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size of the side chain.

Conservative amino acid substitutions generally entail substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Guidance for substitutions, insertions, or deletions can be based on alignments of amino acid sequences of different variant proteins or proteins from different species. Thus, in addition to any naturally-occurring IL-10 polypeptide, the present disclosure contemplates having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 usually no more than 20, 10, or 5 amino acid substitutions, where the substitution is usually a conservative amino acid substitution. [00102] In some cases, IL-10 variant incorporates one or more linkages other than peptide bonds, e.g., at least two adjacent amino acids are joined via a linkage other than an amide bond. For example, in order to reduce or eliminate undesired proteolysis or other means of degradation, and/or to increase serum stability, and/or to restrict or increase conformational flexibility, one or more amide bonds within the backbone of IL-10 can be substituted. In another example, one or more amide linkages (-CO- H-) in IL-10 can be replaced with a linkage which is an isostere of an amide linkage, including but not limited to -CH2 H-, -CH2S-, CH2CH2-, -CH=CH-(cis and trans), -COCH2-, -CH(OH)CH2- or -CH2SO-. One or more amide linkages in IL-10 can also be replaced by, for example, a reduced isostere pseudopeptide bond. See Couder et al. (1993) Int. J. Peptide Protein Res. 41 : 181-184.

[00103] IL-10 variants may comprise one or more non-conservative amino acid substitutions. Examples of non-conservative amino acid substitutions include but are not limited to:

[00104] a) substitution of alkyl-substituted hydrophobic amino acids, including alanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyric acid, (S)-cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from CI -CIO carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions;

[00105] b) substitution of aromatic-substituted hydrophobic amino acids, including phenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine, 1-naphthylalanine,

2naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, including amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy (from ClC4)-substituted forms of the above-listed aromatic amino acids, illustrative examples of which are: 2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4- methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5- methoxytryptophan, 2'-, 3'-, or 4'-amino-, 2'-, 3'-, or 4'-chloro-, 2, 3, or 4-biphenylalanine, 2'-, 3'-, or 4'-methyl-, 2-, 3- or 4biphenylalanine, and 2- or 3-pyridylalanine;

[00106] c) substitution of amino acids containing basic side chains, including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, including alkyl, alkenyl, or aryl -substituted (from CI -CIO branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position. Compounds that serve as illustrative examples include: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3- (4tetrahydropyridyl)-alanine, Ν,Ν-gamma, gamma'-diethyl-homoarginine. Included also are compounds such as alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,

alphamethyl-histidine, alpha-methyl -ornithine where the alkyl group occupies the pro-R position of the alpha-carbon. Also included are the amides formed from alkyl, aromatic, heteroaromatic (where the heteroaromatic group has one or more nitrogens, oxygens or sulfur atoms singly or in combination), carboxylic acids or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives, and lysine, ornithine, or

2,3diaminopropionic acid;

[00107] d) substitution of acidic amino acids, including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of

2,4diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl amino acids;

[00108] e) substitution of side chain amide residues, including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine; and

[00109] f) substitution of hydroxyl -containing amino acids, including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine.

[00110] g) substitution of an amino with a cysteine residue or a cysteine analog can be introduced into an IL-10 polypeptide to provide for linkage to another peptide via a disulfide linkage or to provide for cyclization of the IL-10 polypeptide. Methods of introducing a cysteine or cysteine analog are known in the art; see, e.g., U.S. Patent No. 8,067,532.

[00111] IL-10 variants may also comprise one or more naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids, or D-enantiomers of an amino acid. For example, IL-10 can comprise only D-amino acids. For example, an IL-10 polypeptide can comprise one or more of the following residues: hydroxyproline, β-alanine, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3- diaminopropionic acid, a-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, tbutyl glycine, N-methylisoleucine, phenyl glycine, cyclohexylalanine, norleucine, naphthylalanine, pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine, 2- fluorophenylalanine, 3fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4- tetrahydroisoquinoline-3carboxylic acid, β-2-thienylalanine, methionine sulfoxide,

homoarginine, N-acetyl lysine, 2,4diamino butyric acid, rho-aminophenylalanine, N- methylvaline, homocysteine, homoserine, samino hexanoic acid, co-aminohexanoic acid, co- aminoheptanoic acid, co-aminooctanoic acid, coaminodecanoic acid, co-aminotetradecanoic acid, cyclohexylalanine, α,γ-diaminobutyric acid, a,pdiaminopropionic acid, δ-amino valeric acid, and 2,3-diaminobutyric acid. Additional modifications

[00112] The term IL-10 variant includes a cyclized IL-10 polypeptide. Cyclization may be effected by the introduction of one or more cysteines or cysteine analogs into an IL-10 polypeptide, where the introduced cysteine or cysteine analog can form a disulfide bond with a second introduced cysteine or cysteine analog. Other means of cyclization include introduction of an oxime linker or a lanthionine linker; see, e.g., U.S. Patent No. 8,044,175. Any combination of amino acids (or non-amino acid moieties) that can form a cyclizing bond can be used and/or introduced. A cyclizing bond can be generated with any combination of amino acids (or with an amino acid and -(CH2)n-CO- or -(CH2)n-C6H4-CO-) with functional groups which allow for the introduction of a bridge. Some examples are disulfides, disulfide mimetics such as the -(CH2)n- carba bridge, thioacetal, thioether bridges (cystathionine or lanthionine) and bridges containing esters and ethers. In these examples, n can be any integer, but is frequently less than ten. Other modifications to facilitate cyclization include, for example, an N-alkyl (or aryl) substitution (ψ), or backbone crosslinking to construct lactams and other cyclic structures. Other modifications to facilitate cyclization include C-terminal hydroxymethyl derivatives, o-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides. [00113] The term IL-10 variant includes a retroinverso analog of IL-10 (see, e.g., Sela and Zisman (1997) FASEB J. 11 :449). Retro-inverso peptide analogs are isomers of linear polypeptides in which the direction of the amino acid sequence is reversed (retro) and the chirality, D- or L-, of one or more amino acids therein is inverted (inverso), e.g., using D-amino acids rather than L-amino acids. [See, e.g., Jameson et al. (1994) Nature 368:744; and Brady et al. (1994) Nature 368:692].

[00114] The term IL-10 variant also includes an IL-10 molecules comprising a "Protein

Transduction Domain" or"PTD." A PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. The incorporation of a PTD into a molecule (e.g. IL- 10) facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the amino or carboxy terminus of an IL-10 polypeptide.

[00115] Exemplary protein transduction domains include, but are not limited to, a minimal decapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT); a polyarginine sequence comprising a number of arginine residues sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21 : 1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008), Transportan (as described in Wierzbicki, et al (2014) Folio Histomchemica et Cytobiologica 52(4): 270-280 and Pooga, et a (1998) FASEB J 12(1)67-77 and commercially available from AnaSpec as Catalog No. AS-61256); KALA (as decribed in Wyman et al (1997) Biochemistry 36(10) 3008-3017 and commercially available from AnaSpec as Catalog No. AS- 65459); Antennapedia Peptide (as described in Pietersz et al (2001) Vaccine 19: 1397 and commercially available from AnaSpec as Catalog No. AS-61032); TAT 47-57 (commercially available from AnaSpec as Catalog No. AS-60023) RKKRRQRRR (SEQ ID NO: 15); an arginine homopolymer of from 3 arginine residues to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 16); RKKRRQRR (SEQ ID NO: 17); YARAAARQARA (SEQ ID NO: 18); THRLPRRRRRR (SEQ ID NO: 19); and GGRRARRRRRR (SEQ ID NO: 20).

[00116] The term variant includes modifications to the carboxyl group COR3 of the amino acid at the C-terminal end of an IL-10 polypeptide can be present in a free form (R3 = OH) or in the form of a physiologically-tolerated alkaline or alkaline earth salt including but not limited to, a sodium, potassium or calcium salt. The carboxyl group can also be esterified with primary, secondary or tertiary alcohols including but not limited to, methanol, branched or unbranched Cl-C6-alkyl alcohols, e.g., ethyl alcohol or tert-butanol. The carboxyl group can also be amidated with primary or secondary amines such as ammonia, branched or unbranched C1 -C6- alkylamines or C1-C6 di-alkylamines, e.g., methylamine or dimethylamine.

[00117] The term variant includes modifications to the amino group of the amino acid

NR1R2 at the N-terminus of an IL-10 polypeptide can be present in a free form (Rl = H and R2 = H) or in the form of a physiologically tolerated salt including but not limited to, a chloride or acetate. The amino group can also be acetylated with acids such that Rl = H and R2 = acetyl, trifluoroacetyl, or adamantyl. The amino group can be present in a form protected by amino- protecting groups conventionally used in peptide chemistry, such as those provided above (e.g., Fmoc, Benzyloxy-carbonyl (Z), Boc, and Alloc). The amino group can be N-alkylated in which Rl and/or R2 = C1-C6 alkyl or C2-C8 alkenyl or C7-C9 aralkyl. Alkyl residues can be straight- chained, branched or cyclic (e.g., ethyl, isopropyl and cyclohexyl, respectively).

2. PEGYLATED IL-10

[00118] The present disclosure relates to formulations comprising pegylated IL-10. The terms "pegylated IL-10" and "PEG-IL-10" refer to IL-10 to which one or more polyethylene glycol (PEG) molecules covalently attached to at least one amino acid residue of IL-10. The term "hPEG-IL-10" refers to pegylated human IL-10. Although the method or site of PEG attachment to IL-10 is not critical, in certain embodiments the pegylation does not alter, or only minimally alters, the activity of the IL-10 peptide. In certain embodiments, the increase in half- life is greater than any decrease in biological activity. The biological activity of PEG-IL-10 is typically measured by assessing the levels of inflammatory cytokines (e.g., T Fa or IFNy) in the serum of subjects challenged with a bacterial antigen (lipopolysaccharide (LPS)) and treated with PEG-IL-10, as described in U.S. Pat. No. 7,052,686. The terms "monopegylated IL-10" and "mono-PEG-IL-10" are used interchangeably herein to refer to a PEG-IL10 where one polyethylene glycol molecule is covalently attached to a single amino acid residue of one subunit of the IL-10 dimer. As used herein, the terms "dipegylated IL-10" and "di-PEG-IL-10" are used interchangeably herein to refer to a PEG-ILIO where at least one polyethylene glycol molecule is attached to a single residue on each subunit of the IL-10 dimer. Strategies for pegylation of polypeptides known in the art may be employed in the preparation of PEG-IL10. Pegylation of various molecules is discussed in, for example, U.S. Pat. Nos. 5,252,714; 5,643,575; 5,919,455; 5,932,462; and 5,985,263. Methods for the preparation of PEG-IL10 species are described in United States Patent Nos. 7,052,686 and 8,691,205

[00119] The PEG component of a PEG-IL-10 may have an average molecular weight greater than about 5kDa, greater than about lOkDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 50kDa. In some embodiments, the average molecular weight of the PEG is from about 5kDa to about lOkDa, from about 5kDa to about 15kDa, from about 5kDa to about 20kDa, from about lOkDa to about 15kDa, from about lOkDa to about 20kDa, from about lOkDa to about 25kDa or from about lOkDa to about 30kDa. In the typical practice of the present disclosure, the average molecular weight of the PEG is generally from about 5kDa to about 20kDa. It should be noted that PEG not typically provided as a homogenous preparation of a single PEG species. PEG preparations typically provide PEG molecules having molecular weights over a range as understood by those in the art, a PEG preparation having an average molecular weight of lOkDa is understood by those of skill in the art as comprising multiple PEG species that, on average, have a molecular weight of approximately lOkDa daltons. The PEG can be linear or branched. Branched PEG derivatives, "star-PEGs" and multi -armed PEGs are contemplated by the present disclosure. [00120] The PEG molecule may be covalently attached directly to the IL-10 molecule or may be attached via a polypeptide linker. Suitable linkers include "flexible linkers" which are generally of sufficient length to permit some movement between the modified polypeptide sequences and the linked components and molecules. The linker molecules are generally about 6-50 atoms long. The linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily selected and can be of any suitable length, including but not limited to 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 or more than 50 amino acids. Examples of linkers useful in the practice of the present disclosure include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers and combinations thereof, where m, n, and o are each independently selected from an integer of at least 1 to 20, e.g., 1-18, 216, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10.

[00121] PEG can be bound to a polypeptide of the present disclosure via a terminal reactive group (a "spacer") which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol. The PEG having the spacer which can be bound to the free amino group includes N-hydroxysuccinylimide polyethylene glycol, which can be prepared by activating succinic acid ester of polyethylene glycol with N- hydroxysuccinylimide. Another activated polyethylene glycol which can be bound to a free amino group is 2,4-bis(0-methoxypolyethyleneglycol)-6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride. The activated polyethylene glycol which is bound to the free carboxyl group includes

polyoxyethylenediamine.

[00122] The present disclosure also contemplates compositions of conjugates wherein the PEGs have different n values, and thus the various different PEGs are present in specific ratios. For example, some compositions comprise a mixture of conjugates where n = 1, 2, 3 or 4. In some compositions, the percentage of conjugates where n=l is 18-25%, the percentage of conjugates where n=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and the percentage of conjugates where n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art and provided herein. For example, cation exchange chromatography can be used to separate conjugates, and a fraction is then identified which contains the conjugate having, for example, the desired number of PEGs attached, purified free from unmodified protein sequences and from conjugates having other numbers of PEGs attached.

[00123] For example, the conjugation reaction can be carried out in solution at a pH of from 5 to 10, at temperature from 4°C to room temperature, for 30 minutes to 20 hours, utilizing a molar ratio of reagent to protein of from 4: 1 to 30: 1. Reaction conditions can be selected to direct the reaction towards producing predominantly a desired degree of substitution. In general, low temperature, low pH (e.g., pH=5), and short reaction time tend to decrease the number of PEGs attached, whereas high temperature, neutral to high pH (e.g., pH>7), and longer reaction time tend to increase the number of PEGs attached. Various means known in the art can be used to terminate the reaction. In some embodiments the reaction is terminated by acidifying the reaction mixture and freezing at, e.g., 20°C.

C. Chemotherapeutic Agents:

[00124] Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alky! sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;

ethyl enimines and methyl amelamines including altretamine, triethyienemelamine,

trietylenephosphoramide, triethy!enethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophospharnide, estramustine, ifosfamide, mechlorethanime, mech!orethamine oxide hydrochloride, melphalan, novembichin, phenesteiine, predmmustine, trofosfamide, uracil mustard; mtrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinoniycin, authramycin, azaserine, bleomycins, cactinomycin, caiiclieamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinoniycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5- FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifl uridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutetbimi.de, rnitotane, trilostane; folic acid replenisher such as frolinic acid, aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine;

elfiptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan, lonidamine; niitoguazone, mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide, procarbazine; razoxane; si ofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethyiamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitoiactol; pipobroman; gacytosine; arabinoside (Ara~C); cyclophosphamide, thiotepa; ta oids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;

methotrexate; platinum and platinum coordination complexes such as cisplatin and carbopiatin; vinblastine; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine;

vinoreibine; naveibine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPTI 1 ; topoisomerase inhibitors; difluorom ethyl ornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxyiamoxifen, trioxifene, keoxifene, onapri stone, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, combination therapy comprises administration of a hormone or related hormonal agent. D. Physical Methods

[00125] The compositions of the present disclosure may be administered in combination with anti -neoplastic physical methods such radiotherapy, cryotherapy, hyperthermic therapy, surgery, laser ablation, proton therapy and the like.

IX. Kits

[00126] Kits with unit doses of a subject antibody, e.g. in oral or injectable doses, are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the antibody in treating pathological condition of interest. Examples of compounds and unit doses are those described herein above.

Examples

[00127] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below were performed and are all of the experiments that can be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like described therein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for.

Example 1. Preparation of AM0001 :

[00128] The AM0001 precursor, M9, was generated using Ligand Pharmaceutical's

OmniRat transgenic rats which produce antibodies containing fully human light chains and VH domains, with rat CHI, CH2, and CH3 domains. Conditioned media from the hybridomas was first screened by FACS for binding to human PD-1 recombinantly expressed on the surface of CHO cells. Selected candidates were further screened via FACS for blockade of a PE-labeled PD-Ll-Fc to human PD-1 using the same cell line. The hybridomas from the top 10 candidates were used to express the mAbs and generate purified protein which was further screened by for PD-1 binding in an ELISA format as well as reconfirmation of binding to the human PD-1 CHO cell line. The VH and VL domains of the top 10 candidates were also DNA sequenced. One of the mAbs, M9, exhibited superior binding kinetics to PD-1 compared to the others. AMOOOl is the fully human version of M9 and retains all of the binding and inhibitory characteristics of M9.

Example 2. Inhibition of Binding of PD1 to PDL1 using AMOOOl

[00129] The function of AMOOOl to substantially inhibit the binding of the extracellular domain of PD1 with a PD1 receptor was assessed using Promega' s PD-1 :PD-L1 immune check point assay (Catalog # J1250, J1255) in substantial accordance with the instructions provided by the manufacturer (publications #TM468) . Briefly, the PD-l/PD-Ll Blockade Bioassay is a bioluminescent cell-based assay that can be used to measure the potency and stability of antibodies and other biologies designed to block the PD-l/PD-Ll interaction. The assay consists of two genetically engineered cell lines: PD- 1 Effector Cells, which are Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT response element (NFAT-RE), and; PD-L1 aAPC/CHO-Kl Cells, which are CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate cognate TCRs in an antigen- dependent manner. When the two cell types are co-cultured, the PD- 1/PD-LI interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. Addition of either an anti-PD-1 or anti-PD-Ll antibody that blocks the PD-l /PD-Ll interaction releases the inhibitory signal and results in TCR activation and NFAT-RE-mediated luminescence. The bioluminescent signal can be detected and quantified using a standard luminometer.

[00130] The results of these experiments are summarized in Figure 1. As shown, the these data indicate that AMOOO l 's inhibition of PD-LPD-Ll is comparable to that of both OPDIVO® (nivolumab) and KEYTRUDA® (pembrolizumab). Example 3. Kinetic Studies with AM0001 :

[00131] The affinity of AM0001 was assessed relative to OPDIVO®(nivolumab) and KEYTRUDA® (pembrolizumab) using real time kinetic measurements using an Octet System, (commercially available from Pall ForteBio LLC, 1360 Willow Road, Suite 201, Menlo Park, CA 94025). The data, summarized in Table 5 below, indicate that AMOOOl 's binding affinity for human PD-1 is comparable to that of both OPDIVO® and KEYTRUDA®, products which have demonstrated utility in the treatment of neoplastic disease.

Example 4: Demonstration of AMOOOl Blocking of PPL- 1 Binding

[00132] M9 and AMOOOl exhibit an ECso of 0.35 - 0.5 μg/mL respectively, measured using the Promega immune check point bioassay in substantial accordance with the instructions provided by the manufacturer. This bioassay was prequalified by Promega according to the ICH guidelines and shows the precision, accuracy, and linearity required for routine use in potency and stability studies (PD-1 Technical Manual). The bioassay consists of two genetically engineered cell lines, PD-1 Effector Cells, (Jurkat) and PD-L1 (aAPC/CHO-Kl) antigen presenting cells. When co-cultured, the PD-1/PD-L1 interaction inhibits TCR-mediated luminescence through blockade of nuclear factor of activated T cells (NFAT) activation. When the PD-1/PD-L1 interaction is disrupted, TCR activation induces luminescent through the activation of NFAT. This luminescence can be detected by addition of Bio-Glo™ Reagent and quantified with a luminomitor. The results of these studies are presented in Figure 2 of the attached drawings illustrating that AMOOOl blocks PDL1 binding. Example 5. Binding Specificity of AMOOOl

[00133] AMOOOl binding specificity was tested in an ELISA format by binding to human, cynomolgus monkey, murine, rat, and canine PD-l-Fc fusion proteins, with detection by anti- human IgG:HRP. The results of these studies are provided in Figures 3, 4, and 5 of the attached drawings. As illustrated, AMOOOl binds to both human and cynomologus monkey (Figure 3) but not murine (Figure 3), rat (Figure 4), or canine (Figure 5) PD-1.

Example 6. AMOOOl Binding to Deglycosylated PDl-Fc

[00134] Human and mouse PD-l-Fc proteins were deglycosylated with PNGase F as detailed in Table 6.

[00135] The deglycosylation reactions were sampled at various intervals via Coomassie (4-12% Bis-Tris/MES reducing) to verify complete deglycosylation (Figure 6). Reactions were stopped at 25 hours and used to coat a Nunc MaxiSorp™ high binding 96-well ELISA plate (VWR Scientific) overnight at 4°C at lug/ml, and lOOul/well, 2 plates). The following day the plate was blocked with ELISA Blocker Blocking Buffer (Catalog No. 502, Thermo Fisher), 200ul/well for 2 hours at room temperature (approximately 25 °C) with rocking. The coated plate was then probed with AMOOOl or nivolumab (lOmg/ml, serially diluted 1 :2 in PBS down 16 wells to a final volume of lOOul/well) for one hour at room temperature with rocking.

Binding to the glycosylated and deglycosyalted PD1-FC was detected using a goat anti-human IgG:HRP (Abeam, 1 : 10,000 in PBS, lOOul/well, 1 hour at room termperature, with rocking), and developed with 1-Step Ultra TMB-ELISA solution (Therm oFisher, Catalog number: 34028), lOOul/well for approximately 3 minutes at room temperature. The reaction was quenched by the addition lOOul/well of Stop Solution (Life Technologies). The plates were read on a Molecular Devices SpectraMax 340PC plate reader (A450nm, Automix on). The results are presented in Figure 7 of the attached drawings. As illustrated in Figure 7, the binding of AMOOOl 's is affected by PD-1 glycosylation in contrast to the binding of nivolumab which is not affected by PD-1 glycosylation (Figure 7) indicating that the AMOOOl and Nivolumab do not bind to the same epitope.

Example 7. AMOOOl Blocks PD-1/PD-L1 and PD-L2 Binding

[00136] Using the Octet system, it was investigated whether AMOOOl blocked the interaction of PD-1 with PD-L1 or PD-L2. In this experiment, PD-L1 or PD-L2:Fc fusion proteins were bound to the sensor. A threefold molar excess of AMOOOl was pre-incubated with PD-1 :Fc fusion protein and then assessed for affinity. Figure 8 below illustrate that

pre-incubation of AMOOOl with PD-1 prior to exposure to PD-L1 or PD-L2 bound to the sensor blocks the interaction with PD-L1 or PD-L2, respectively.

Example 8: AMOOOl Blockade of endogenous PD-1 function

[00137] AMOOOl 's capacity to inhibit endogenous PD-1 function was assessed in the context of normal peripheral blood mononuclear cells response to staphylococcal enterotoxin B (SEB). A modified assay using whole blood instead of peripheral blood mononuclear cells (PBMC) (Wang, et al (2014) Cancer Immunology Research 2(9):846-56) was used. Briefly, whole blood was diluted 10-fold with complete Roswell Park Memorial Institute (RPMI) medium, and exposed to SEB with or without AMOOOl . After 3 to 4 days incubation, the cells were pelleted by centrifugation and the supernatant was measured by ELISA for IL-2. The results are presented in Figure 9 of the accompanying drawings and illustrates a donor response to SEB with an increasing titration of AMOOOl . These data are representative of 4 normal donors' response to PD-1 inhibition with AMOOOl.

Claims

CLAIMS I claim:

1. A PD1 polypeptide binding molecule ("PDl-PBM") comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

2. The PDl-PBM of claim 1 wherein said PDl-PBM binds to the extracellular domain of a mammalian PD1.

3. The PDl-PBM of claim 1 or 2 wherein said PDl-PBM comprises amino acid sequences of SEQ ID NOS: 1 - 6.

4. The PDl-PBM of any one of claims 1-3, wherein said PDl-PBM comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of

DISGGGGTTYYADSVKG (SEQ ID NO:5), and a CDR3 comprising the amino acid sequence of SGTVVTDFDY (SEQ ID NO:6).

5. The PDl-PBM of any one of claims 1-4, wherein said PDl-PBM comprises a light chain variable region comprising a CDR1 comprising the amino acid sequence of GGNSIGSYSVH (SEQ ID NO: 1), a CDR2 comprising the amino acid sequence of DDSDRPS (SEQ ID NO: 2), and a CDR3 comprising the amino acid sequence of QVWDTSSYWV (SEQ ID NO: 3).

6. The PDl-PBM of claim 5, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

7. The PDl-PBM of any one of claims 1-6, wherein said PDl-PBM comprises a mammalian IgG framework.

8. The PDl-PBM of claim 7, wherein said IgG framework is IgGl, IgG2, IgG3 or IgG4.

9. The PDl-PBM of claim 7, wherein said mammalian IgG framework is derived from a human IgGl, IgG2, IgG3 or IgG4 monoclonal antibody.

10. The PDl-PBM of claim 7, wherein said mammalian IgG framework is an IgG4 framework.

11. The PDl-PBM of claim 10, wherein the IgG4 framework is derived from an anti-PDl IgG4 monoclonal antibody selected from the group consisting of nivolumab, and pembrolizumab, lambrolizumab.

12. The PDl-PBM of claim 7 wherein said mammalian IgG framework is an IgGl framework.

13. The PDl-PBM of claim 7 wherein said mammalian framework is an IgG4 framework.

14. The PDl-PBM of any of claims 1-13, wherein said PDl-PBM has an affinity for the

extracellular domain of PD1 of having a Kd of less than 10e-12 and substantially inhibits the ability of said extracellular domain of a mammalian PD1 to bind to one or more PD1 ligands.

15. A pharmaceutical composition comprising a PDl-PBM of any of claims 1-14 and a

pharmaceutically acceptable carrier.

16. A recombinant mammalian cell comprising a nucleic acid comprising a nucleotide sequence encoding a heavy chain polypeptide and a nucleic acid comprising a nucleotide sequence encoding a light chain, wherein the heavy and light chains are the heavy and light chains of the PD 1 -PBM of any of claims 1 - 13.

17. The recombinant mammalian cell of claim 16, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 8.

18. The recombinant mammalian cell of claim 16 or 17, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 7.

19. An expression vector comprising:

a nucleic acid comprising a nucleotide sequence encoding a heavy chain polypeptide; and/or a nucleic acid comprising a nucleotide sequence encoding a light chain,

wherein the heavy and light chains are the heavy and light chain polypeptides of the PDl- PBM of any of claims 1-13.

20. The expression vector of claim 19, wherein the heavy chain comprises the amino acid

sequence of SEQ ID NO: 8.

21. The expression vector of claim 19 or 20, wherein the light chain comprises the amino acid sequence of SEQ IDNO: 7.

22. A recombinant host cell comprising an expression vector of any of claims 19-21.